Growing living cells in vitro can be performed for a variety of purposes, including the production of cell derivatives and secreted products, the preparation of viral vaccines, expansion and harvesting of the cells themselves, and the recovery of valuable cell by-products. There are a variety of methods used for cell culture at the production level. These can be as simple as banks of roller bottles, disposable bags on a rocking platform, to large stirred vessels having a volume of 10,000 liters or more.
These systems suffer from certain shortcomings. For example, cells bound to a non-porous surface must be split prior to the cells reaching confluence. Adherent cells need to be adapted to suspension culture for production in large tanks. The larger the volume of the reactor, the greater the potential for local variability of cell culture conditions within the system. Also, the volume of cell culture product to be processed can be quite large and the concentration of the desired product can be quite low.
Among the devices that have been developed for growing cells in vitro, the shell-and-tube type arrangement has become fairly common, particularly for growing suspension and adherent cells. Such devices, which can also be referred to as hollow-fiber bioreactors, use permeable tube-shaped hollow fibers (e.g., capillaries), contained within an outer shell, which may be configured so that fluid in a space external to the hollow fibers (an extra-capillary space) is segregated from lumens of the hollow fibers and fluid passing therethrough. Additionally, such devices typically include two manifold end chambers within the outer shell located at opposite ends of the device. Each end of the lumen of a hollow fiber connects to a different end chamber. The end chambers and the fiber lumens are separated from the extra-capillary space by the permeable membranes of the hollow fibers. Transport of aqueous and/or gaseous substances between the fiber lumens and the extra-capillary space can be controlled, to a certain extent, by the molecular weight cutoff, or pore size, of the membranes of the hollow fibers.
Typically, cells in a hollow-fiber bioreactor are grown in the extra-capillary space while a nutrient medium is passed through the hollow fibers. The permeable nature of the hollow fibers allows nutrients and cell waste products and/or by-products to pass through the walls of the hollow fibers while blocking cells from doing the same. For example, U.S. Pat. No. 4,391,912 to Yoshida et al. describes a range of pore diameters to support the transfer of the nutrient medium from the intra-capillary space (e.g., within the lumen of a fiber) to the extra-capillary space, while blocking the entrance of cells into the intra-capillary space.
Shell-and-tube type bioreactors provide several advantages. For adherent cells, the use of several hollow fibers can provide a large amount of surface area upon which the cells can grow within a relatively small reactor volume. For both suspension and adherent cells, this large surface area density can facilitate localized distribution of nutrient media to the growing cells and collection of cell waste products. Hollow-fiber bioreactors thus may enable the growth of cells at much higher densities than is possible with many other cell culture devices. For example, they can support cell densities greater than 10^8 cells per milliliter, whereas other cell culture devices are typically limited to densities around 10^6 cells per milliliter. This high cell density facilitates the adaptation of the cells to a simplified serum free medium.
U.S. Pat. No. 6,933,144 of Cadwell describes a hollow-fiber bioreactor that includes a hollow fiber cartridge provided between two deformable bags, where providing nutrients to cells in the extra-capillary volume can be achieved through bi-directional flow of liquid medium between the two bags as they are raised and lowered. The force of gravity impels medium flow through the lumens of the capillaries. Very high flow rates can be achieved through this system and method, which is shown in FIGS. 1-3 and described below.
FIG. 1 shows a cross sectional side view of a prior-art hollow-fiber bioreactor. In this bioreactor, a media reservoir 102 holds cell-culture media 104 and is configured to be rocked or rotated about a horizontal axis of rotation 106 that extends into the drawing sheet of FIG. 1. An enclosed chamber 108 is disposed within the media reservoir 102, wherein an extra-chamber space 110 is defined between the media reservoir 102 and the enclosed chamber 108. A plurality of hollow fibers 112 pass through the enclosed chamber 108 and are secured at each end by a first potting structure 114 and a second potting structure 116. An extra-capillary space 118 is defined between an interior of the enclosed chamber 108 and the exterior surfaces of the hollow fibers 112. For example, the fibers 112 are potted at the ends of the chamber 108 such that any liquid media 104 entering the end of the chamber 108 passes through the fiber lumens and out the other end of the chamber 108, such that no media 104 directly enters the extra-capillary space 118 as it flows through the reactor, but remains separated from the extra-capillary space 118 by the walls of the fibers 112. The hollow fibers 112 are oriented substantially parallel to a longitudinal axis 120, which can be substantially perpendicular to the horizontal axis of rotation 106.
The media reservoir 102 can include an opening 122 for accessing the extra-chamber space 110, e.g., to allow fresh cell-culture media to be supplied to the media reservoir 102, to allow stale cell-culture media to be removed from the media reservoir 102, and/or to facilitate removal of cell waste products from the media reservoir 102. A lid 124 can be provided to seal the opening 122. The media reservoir 102 can also include one or more openings 126 for accessing the extra-capillary space 118. For example, the opening 126 includes a port 128 passing through the extra-chamber space 110 to provide access to the extra-capillary space 118. The opening 126 allows developing cells to be placed into extra-capillary space 118, mature cells to be removed from the extra-capillary space 118, secreted products to be harvested from the extra-capillary space 118, and/or administration of reagents, drugs, and/or DNA or RNA vectors to the cells.
The media reservoir 102 can also include a gas-permeable membrane 130 permitting gas exchange between an environment exterior to the media reservoir 102 and the extra-chamber space 110. The membrane 130 permits the exchange of the waste gases from the extra-chamber space 110 with fresh gases from the environment exterior to the media reservoir 102. Transverse members 134 provide support to the media reservoir 102 along a face that includes membrane 130. A dam 136 is disposed in the media reservoir 102 to impede flow of cell-culture media 104 within the extra-chamber space 110 when the media reservoir 102 is rocked or rotated about the horizontal axis of rotation 106. The dam 136 also serves to encourage flow of cell-culture media 104 through the hollow fibers 112. If the enclosed chamber 108 spans the width of the media reservoir 102 along the horizontal axis of rotation 106, the enclosed chamber 108 and dam 136 can be integrated.
FIG. 2 is a cutaway end view showing a partial cross section of the hollow-fiber bioreactor shown in FIG. 1 taken along section A-A′. In FIG. 2, each hollow fiber 112 has a central lumen 202 that is open at each end to the extra-chamber space 110, such that cell-culture media 104 can pass through the lumens 202 of hollow fibers 112, e.g., to facilitate passage of nutrients through the walls of hollow fibers 112 to nourish the cells in the extra-capillary space 118.
FIG. 3 illustrates how a rocking or rotating motion causes the flow of cell-culture media in the hollow-fiber bioreactor shown in FIG. 1. By impeding the flow of cell-culture media 104 in the extra-chamber space 110, the dam 136 simultaneously increases the static head pressure of a raised portion 302 of cell-culture media 104 and decreases the static head pressure of a lowered portion 304 of cell-culture media 104 that would otherwise exist in the absence of dam 134. Thus, by increasing the differential pressure across the hollow fibers 112, the dam 136 serves to encourage flow of cell-culture media 104 through the hollow fibers 112 when the reactor is tilted around the axis 106.
The length of time the cells can be cultured in a conventional hollow-fiber bioreactor may be extended to many months, such that scale up of production can be achieved by longer culture times rather than by using different equipment. However, it is generally recognized that the delivery of oxygen to cells growing in these systems can present a limitation to the size of hollow fiber bioreactors. This is primarily due to the low solubility of gases such as oxygen and carbon dioxide in aqueous solutions at the temperatures required for cell culture, and limitations of flow rate generated by such bioreactor systems. This physicochemical phenomenon has an impediment to the creation and adoption of larger scale hollow fiber cell culture systems.
Insufficient oxygenation represents a primary shortcoming of conventional hollow fiber bioreactor systems, and is the primary reason that the technology is underutilized and cannot be used at a scale practicable for larger scale bio-manufacturing despite its many advantages. There are examples in the prior art that attempt to address these issues, without particular success. For example, gas exchange can be accomplished by passing the medium through a device that passively diffuses gas into the medium prior to entering the hollow fiber cartridge, where such gases may then pass (in a limited fashion) through the walls of the fibers and into the extra-capillary volume where the cells are located. An example of this approach is described in US Patent Publication No. 2010/0159524 of Smith et. al. A bioreactor system that uses two fiber types within the cartridge, one to deliver a nutrient medium and one to deliver gas, is described in U.S. Pat. No. 5,622,857 of Goffe, and in U.S. Pat. No. 6,680,166 of Mullon et. al. Another type of bioreactor, described in U.S. Pat. No. 6,979,308 of MacDonald et al., includes concentric hollow fibers of increasing diameter, one within the other. Within the spaces defined by the hollow fibers are defined spaces for medium flow, gas delivery, and cell growth. Such reactors deigned to improve gas delivery can be complex in design and operation, and may further be limited in the amount of gases that can be delivered to the cells in the extra-capillary space.
Therefore, there may be a need to provide method, device and/or apparatus that can provide improved transfer and control of gases and media to cells in a bioreactor. These and other objects, features and advantages of the present disclosure will become apparent upon reading the following detailed description of exemplary embodiments of the disclosure.